Apparatus for electroporation mediated delivery for drugs and genes

ABSTRACT

A device for in vivo electroporation therapy comprising an electrode applicator with at least two pairs of electrodes arranged relative to one another to form an array and a power supply. The device is used to generate an electric field in a biological sample and effect introduction of selected molecules into cells of the sample.

This application is a continuation of and claims the benefit under 35USC § 120 of prior U.S. application Ser. No. 09/861,016, filed May 18,2001, now U.S. Pat. No. 6,516,223 which is a continuation of Ser. No.09/177,678, filed Oct. 22, 1998, issued Jun. 5, 2001 as U.S. Pat. No.6,241,701; which is a continuation-in-part of U.S. application Ser. No.08/905,240, filed Aug. 1, 1997, issued Apr. 25, 2000 as U.S. Pat. No.6,055,453, all of the disclosures of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the use of electric pulses toincrease the permeability of cells, and more specifically to a methodand apparatus for the application of controlled electric fields for invivo delivery of pharmaceutical compounds and genes into cells byelectroporation therapy (EPT), also known as cell poration therapy (CPT)and electrochemotherapy (ECT).

2. Description of the Related Art

In the 1970's it was discovered that electric fields could be used tocreate pores in cells without causing permanent damage. This discoverymade possible the insertion of large molecules into cell cytoplasm. Itis known that genes and other molecules such as pharmacologicalcompounds can be incorporated into live cells through a process known aselectroporation. The genes or other molecules are mixed with the livecells in a buffer medium and short pulses of high electric fields areapplied. The cell membranes are transiently made porous and the genes ormolecules enter the cells, where they can modify the genome of the cell.

Electroporation in vivo is generally limited to tissue or cells that areclose to the skin of the organism where the electrodes can be placed.Therefore, tissue which would otherwise be treatable by systemic drugdelivery or chemotherapy, such as a tumor, is generally inaccessible toelectrodes used for electroporation. In the treatment of certain typesof cancer with chemotherapy, it is necessary to use a high enough doseof a drug to kill the cancer cells without killing an unacceptable highnumber of normal cells. If the chemotherapy drug could be inserteddirectly inside the cancer cells, this objective could be achieved. Someof the anti-cancer drugs, for example, bleomycin, normally cannotpenetrate the membranes of certain cancer cells effectively. However,electroporation makes it possible to insert bleomycin into cells.

Treatment typically is carried out by injecting an anticancer drugdirectly into the tumor and applying an electric field to the tumorbetween a pair of electrodes. The field strength must be adjustedreasonably accurately so that electroporation of the cells of the tumoroccurs without damage, or at least minimal damage, to any normal orhealthy cells. This can normally be easily carried out with externaltumors by applying the electrodes to opposite sides of the tumor so thatthe electric field is between the electrodes. When the field is uniform,the distance between the electrodes can then be measured and a suitablevoltage according to the formula E=V/d can then be applied to theelectrodes (E=electric field strength in V/cm; V=voltage in volts; andd=distance in cm). When large or internal tumors are to be treated, itis not easy to properly locate electrodes and measure the distancebetween them. The aforementioned parent application discloses a systemof electrodes for in vivo electroporation wherein the electrodes may beinserted into the tumor. In related U.S. Pat. No. 5,273,525, a syringefor injecting molecules and macromolecules for electroporation utilizesneedles for injection which also function as electrodes. Thisconstruction enables subsurface placement of electrodes.

Treatment of a subject using cell poration therapy provides a means foravoiding the deleterious effects typically associated withadministration of anticancer or cytotoxic agents. Such treatment wouldallow introduction of these agents to selectively damage or killundesirable cells while avoiding surrounding healthy cells or tissue.

SUMMARY OF THE INVENTION

It is a primary object of the invention to provide an improved apparatusthat can be conveniently and effectively positioned to generatepredetermined electric fields in pre-selected tissue.

In accordance with a primary aspect of the invention, an electrodeapparatus for the application of electroporation to a portion of thebody of a patient comprises a support member, a plurality of needleelectrodes mounted on said support member for insertion into tissue atselected positions and distances from one another, and means including asignal generator responsive to said distance signal for applying anelectric signal to the electrodes proportionate to the distance betweensaid electrodes for generating an electric field of a predeterminedstrength.

The invention includes needles that function for injection oftherapeutic substances into tissue and function as electrodes forgenerating electric fields for portion of cells of the tissue.

One embodiment of the invention includes a system for clinicalelectroporation therapy that includes a needle array electrode having a“keying” element, such as a resistor or active circuit, that determinesthe set point of the therapy voltage pulse, as well as selectable arrayswitching patterns (the apparatus having this system has been termedMedPulser™). A number of electrode applicator designs permit access toand treatment of a variety of tissue sites.

Another embodiment of the invention provides a laparoscopic needleapplicator that is preferably combined with an endoscope for minimallyinvasive electroporation therapy.

The invention provides a therapeutic method utilizing the needle arrayapparatus for the treatment of cells, particularly tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-section assembly drawing showing a view of anembodiment of the invention.

FIGS. 2 a-2 g are diagrammatic illustrations of several alternativeelectrode embodiments in accordance with the invention.

FIG. 3 is a block diagram of a treatment instrument in accordance withthe invention.

FIGS. 4A-C are a schematic block diagram of the circuitry for thetreatment instrument of FIG. 3.

FIG. 5 is a schematic diagram of selector switching elements of thecircuit shown in FIG. 4.

FIG. 6 diagrammatically shows a preferred 4×4 mapping array for needlesforming 9 treatment zones in accordance with one embodiment of theinvention.

FIG. 7 a shows a pulse sequence for a 2×2 treatment zone in accordancewith one embodiment of the invention.

FIGS. 7 b-7 d shows a pulse sequence for a 6 needle array in accordancewith one embodiment of the invention.

FIG. 8 is a diagram of a prior art endoscopic examination system.

FIGS. 9 a-9 b show in detail an extending/retracting needle array inaccordance with the invention.

FIG. 10 shows the tumor volume up to 120 days of EPT with bleomycin inPanc-3 xenografted nude mice (D=drug; E=electroporation), for the venouscontrol groups (D+E−, D−E−, D−E+), and the treated group (D+E+).

FIGS. 11 a and 11b show the effect of EPT of Panc-3 withneocarcinostatin for the pre- and post-pulse injection of the drug,respectively, up to day 24.

FIG. 12 shows the tumor volume after 34 days of EPT with bleomycin innon-small cell lung carcinoma (NSCLC) xenografted nude mice. The arrowindicates retreatment of one mouse at day 27 (D=drug;E=electroporation).

FIGS. 13 a-13 d show the sequences of events involved in the treatmentof the tumor xenograft (a) by EPT. The treatment led to the formation ofa scar (b) which dried and ultimately fell off (c) leaving a clearhealed area of skin (d) free of tumor.

FIGS. 14 a-14 c show the histology of tumor samples carried out 35 daysafter the treatment. D+E+ group shows necrotic tumor cell ghosts (b)compared to a mixture of viable and nectrotic cells in D+E− group (a).Histology of samples from tumor site after 120 days show completeabsence of tumor cells (c).

FIGS. 15 a and 15 b show the survival of MCF-7 (breast cancer) cellswhen exposed to low voltage and high voltage EPT, respectively.

FIGS. 16 a and 16 b show the survival of MCF-7 cells when exposed to lowvoltage and high voltage EPT, respectively, with bleomycin.

FIG. 17 shows the effect of non-pulsed and pulsed MCF-7 cells withdifferent concentration of bleomycin and the MedPulser™.

FIGS. 18 a and 18 b show the invention electrode applicator including ashield element that separates the applicator handle from the electrodeapplicator. FIG. 18 a shows the electrode applicator in perspectiveview. FIG. 18 b shows an exploded lateral cross-section of the inventionelectrode applicator.

FIG. 19 shows a support member of the invention apparatus having aplurality of needle electrodes mounted thereon for insertion intotissue.

Like reference numbers and designations in the various drawings indicatelike elements.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Overview

The invention provides an apparatus and a method for the therapeuticapplication of electroporation. The method includes injection of achemotherapeutic agent or molecule and electroporation of the agent ormolecule into a tumor. In particular, an agent or molecule is injectedinto tissue and voltage pulses are applied between “needle” electrodesdisposed in the tissue, thus applying electric fields to cells of thetissue. The needle electrode assemblies described below enable the invitro or in vivo positioning of electrodes in or adjacent to subsurfacetumors or other tissue. Such therapeutic treatment is calledelectroporation therapy (EPT), also called electrochemotherapy. Whilethe focus of the description below is EPT, the invention may be appliedto other treatments, such as gene therapy of certain organs of the body.

For a general discussion of EPT, see co-pending application Ser. No.08/537,265, filed on Sep. 29, 1995, which is a continuation-in-part ofapplication Ser. No. 08/467,566 filed on Jun. 6, 1995, which is acontinuation-in-part of application Ser. No. 08/042,039 filed on Apr. 1,1993 now abandoned, all of which are incorporated herein by reference.

Electrode Assemblies

FIG. 1 is a cross-section assembly drawing showing a view of a needleassembly 100 in accordance with one embodiment of the invention. Theneedle assembly 100 comprises an elongated tubular support body or shaft112, which may be hollow stainless steel or a medical-grade plastic(e.g., nylon). If the shaft is made of a conductive material, electricalinsulation should be applied on the exterior services to protect bothpatient and physician. The shaft 112 includes a plurality of electrodeneedles 114 at the distal end, coupled to respective conductors of amulti-conductor wire cable 116. The electrode needles 114 may be sharpor blunt, hollow or solid, and of any desired length. The material ofthe electrode needles 114 must be electrically conductive, but need notbe a metal or uniform (i.e., a composite or layered structure may beused, such as metal-coated plastic or ceramic needles). One or morehollow electrode needles 114 may be used to inject a therapeuticsubstance. In different embodiments, the electrode needles 114 maycomprise a rectangular, hexagonal, or circular array. However, otherpatterns may be used.

In use, the multi-conductor wire cable 116 is coupled to a high-voltagegenerator. In the illustrated embodiment, a retractable shield 118,restricted by a friction O-ring 120 near the distal end can be slidefore and aft along the shaft 112 body to protect or expose the electrodeneedles 114.

FIGS. 2 a-2 e are diagrammatic illustrations of several alternativeelectrode embodiments in accordance with the invention. FIGS. 2 a and 2b show straight-bodied electrodes having needles 200 with differentspacing. For example, the needles in FIG. 2 a comprise a 0.5 cm diameterarray, while the needles in FIG. 2 b comprise a 1.4 cm diameter array.The various body dimensions may vary as well. For example, the electrodein FIG. 2 a has a stepped body structure, with a smaller diameterfore-body 202 relative to a larger diameter aft-body 204. The electrodein FIG. 2 b has a uniform diameter body 206. The electrodes in FIGS. 2 aand 2 b are particularly well suited for treating small surface tumors.

FIGS. 2 c and 2 d show angled-head electrodes having needle tips 200 setat an angle with respect to the bodies 206 of the electrodes. FIG. 2 cshows the needle-tips at about a 45° angle with respect the body 206.FIG. 2 d shows the needle-tips at about a 90° angle with respect thebody 206. The electrodes in FIGS. 2 c and 2 d are particularly wellsuited for treating head and neck tumors.

FIG. 2 e shows a double-angled electrode having needle tips 200 set atan angle with respect to a smaller diameter fore-body 202. A largerdiameter aft-body 204 is angled as well. The electrode in FIG. 2 e isparticularly well suited for treating tumors of the larynx, but may alsobe used in other body cavities.

FIG. 2 f shows an electrode particularly well suited for treating largetumors. The spacing between needles 208 may be, for example, about 0.65cm. FIG. 2 g shows an electrode particularly well suited for treatinginternal tumors. The spacing between needles 208 may be, for example,about 1.0 cm.

Any of the separate configuration elements (e.g., body size andconfiguration, head and body angle, etc.) shown in FIGS. 2 a-2 g can becombined as desired. Other configurations of electrode assemblies may beused to meet particular size and access needs.

EPT Instrument

FIG. 3 is a diagram of an EPT treatment instrument 300 embodying theinvention. An electrode applicator 312 is removably coupled to theinstrument 300, which selectively applies voltage pulses to selectedelectrode needles 314 of the electrode applicator 312. The pulseduration, voltage level, and electrode needle addressing or switchingpattern output by the instrument 300 are all programmable.

A display 316 indicates the therapy voltage setpoint. A remote therapyactivation connection 318 is provided to a accommodate a foot pedalswitch 320 for activating pulses to the electrode applicator 312. Thefoot pedal switch 320 permits a physician to activate the instrument 300while freeing both hands for positioning of the electrode applicator 312in a patient's tissue. Indicator lights 322 for fault detection, poweron, and completion of a therapy session are provided for convenience.Other indicator lights 324 are provided to positively indicate that anelectrode applicator 312 is connected to the instrument 300 and toindicate the type of needle array (see discussion below). Astandby/reset button 326 is provided to “pause” the instrument and resetall functions of the instrument to a default state. A ready button 328is provided to prepare the instrument 300 for a therapy session. Aprominent “therapy in process” indicator light 330 indicates thatvoltage pulses are being applied to the electrode needles 314. Inaddition, the instrument 300 may have audio indicators for suchfunctions as a button press, a fault state, commencement or terminationof a therapy session, indication of therapy in process, etc.

In an alternative embodiment, the instrument 300 can be coupled to afeedback sensor that detects heart beats. Applying pulses near the heartmay interfere with normal heart rhythms. By synchronizing application ofpulses to safe periods between beats, the possibility of suchinterference is reduced.

FIG. 4 is a schematic block diagram of the circuitry 400 for thetreatment instrument 300 of FIG. 3. An AC power input module 402provides electrically isolate power for the entire instrument 300. Alow-voltage DC power supply 404 provides suitable power for the controlcircuitry of the instrument 300. A high-voltage power supply 406provides suitable high voltages (e.g., up to several thousand volts)needed for EPT therapy. The output of the high-voltage power supply 406is coupled to a pulse power assembly 408 which generates pulses ofvariable width and voltage under control from a controller assembly 410.The output of the pulse power assembly 408 is coupled through a highvoltage switch array 412 to a needle array connector 414. A remotetherapy activation foot peddle connector 416 permits attachment of afoot pedal switch 320.

The high voltage switch array 412 allows the necessary high voltages forEPT to be applied to selected subgroups of electrodes in a needleassembly 100. In prior versions of EPT instruments, application of suchvoltages has typically involved use of a manual rotary “distributor”switch, or a motorized version of such a switch. However, in the presentinvention, all switching is by electronically controlled relays,providing for faster and quieter switching, longer life, and better andmore flexible control over switching patterns.

FIG. 5 is a schematic diagram of one selector switching element 500 ofthe high voltage switch array 412 of the circuit shown in FIG. 4. Thenumber of such switching elements 500 should at least match the largestnumber of electrodes of any attached needle assembly 100. Each switchingelement 500 provides for control of the high-voltages applied to anelectrode of a needle assembly 100, with the ability to provide voltageat either polarity to the associated electrode.

In particular, when a “negative” control voltage is applied to oneinverting input amplifier 502 a, an associated, normally open relay 504a is closed, establishing a negative return path for a pulse applied toa paired electrode to be coupled through an electrode connector 506.Similarly, when a “positive” control voltage is applied to a secondinverting input amplifier 502 b, an associated, normally open relay 504b is closed, establishing a path for a positive pulse to be applied toan electrode coupled through the electrode connector 506.

Needle Array Addressing

The instrument 300 of FIG. 3 is designed to accommodate electrodeapplicators 312 having varying numbers of electrode needles 314.Accordingly, an addressing scheme has been developed that, in thepreferred embodiment, permits addressing up to 16 different needles,designated A through P, forming up to 9 square treatment zones andseveral types of enlarged treatment zones. A treatment zone comprises atleast 4 needles in a configuration of opposing pairs that are addressedduring a particular pulse. During a particular pulse, two of the needlesof a treatment zone are of positive polarity and two are of negativepolarity.

FIG. 6 diagrammatically shows a preferred 4×4 mapping array for needlesforming 9 square treatment zones numbered from the center and proceedingclockwise. In the preferred embodiment, this mapping, array defines4-needle, 6-needle, 8-needle, 9-needle, and 16-needle electrodeconfigurations. A 4-needle electrode comprises needles placed inpositions F, G, K, and J (treatment zone 1). A 9-needle electrodecomprises needles placed in positions defining treatment zones 1-4. A16-needle electrode comprises needles placed in positions definingtreatment zones 1-9.

FIG. 7 a shows a pulse sequence for a 2×2 treatment zone in accordancewith one embodiment of the invention. During any of four pulsescomprising a cycle, opposing pairs of needles are respectivelypositively and negatively charged, as shown. Other patterns of suchpairs are possible, such as clockwise or counterclockwise progression.For a 9-needle electrode configuration, a preferred cycle comprises 16pulses (4 treatment zones at 4 pulses each). For a 16-needle electrodeconfiguration, a preferred cycle comprises 36 pulses (9 treatment zonesat 4 pulses each).

A 6-needle electrode configuration can comprise a circular or hexagonalarray as shown in FIGS. 7 b-7 d. Alternatively, a 6-needle electrodeconfiguration can be defined as a subset of a larger array, such as isshown in FIG. 6. For example, with reference to FIG. 6, a 6-needleelectrode configuration can be defined as a 2×3 rectangular array ofneedles placed in positions defining treatment zones 1-2 (or any otherlinear pair of treatment zones), or a hexagonal arrangement of needlesB, G, K, N, I, E (or any other set of positions defining a hexagon)defining an enlarged treatment zone (shown in dotted outline in FIG. 6).Similarly, an 8-needle electrode can comprise an octagon, or a subset ofthe larger array shown in FIG. 6. For example, with reference to FIG. 6,an 8-needle electrode can be defined as a 2×4 array of needles placed inpositions defining treatment zones 1, 2 and 6 (or any other lineartriplet of treatment zones), or an octagonal arrangement of needles B,C, H, L, O, N, I, E (or any other set of positions defining an octagon)defining an enlarged treatment zone.

FIGS. 7 b-7 d shows a hexagonal arrangement and one possible activationsequence. FIG. 6 b shows a first sequence, in which needles G and K arepositive and needles I and E are negative during a first pulse, and havereversed polarities during a next pulse; needles B and N, shown indotted outline, are inactive. FIG. 6 c shows a second sequence, in whichneedles K and N are positive and needles E and B are negative during afirst pulse, and have reversed polarities during a next pulse; needles Gand I are inactive. FIG. 6 de shows a third sequence, in which needles Nand I are positive and needles B and G are negative during a firstpulse, and have reversed polarities during a next pulse; needles K and Eare inactive. A total of 6 pulses are applied in a cycle of sequences. Asimilar activation sequence can be used for an octagonal arrangement.

Regardless of physical configuration, the preferred embodiments of theinvention always uses at least two switched pairs of electrodes (forexample, as shown in FIG. 7 a) in order to achieve a relatively uniformelectric field in tissue undergoing EPT. The electric field intensityshould be of sufficient intensity to allow incorporation of a treatmentagent in order to effect the process of electroporation.

Automatic Identification of Electrode Applicators

The mapping scheme described above permits different electrodeapplicators 312 to be coupled to the same instrument 300. Since thenumber of electrode needles 314 can vary, the invention includes a meansfor automatically configuring the instrument 300 to address the propernumber of electrode needles 314. In one embodiment, each electrodeapplicator 312 includes a built-in type identification element, such asa “keying” resistor, that permits the instrument 300 to determine thenumber of electrode needles 314, and thus set itself to a matchingaddressing scheme. The instrument 300 reads the type identificationelement when an electrode applicator 312 is coupled to the instrument300. The type identification element may be incorporated into aconnector for the electrode applicator 312 and access through shared ordedicated electrical connections.

As an illustrative example, the following table maps resistor values tothe number of electrode needles 314:

Needle Array Type ID Resistor (ohms) Needle Addressing Scheme 787 6 4536 232 6 4.32K 9 2.21K 16  1.29K 16 

A similar technique can be used to automatically set the therapy voltagefor the instrument 300. That is, each electrode applicator 312 includesa built-in voltage identification element, such as a “keying” resistor,that permits the instrument 300 to determine the proper voltage levelfor treatment pulses for the particular electrode applicator 312. Theinstrument 300 reads the voltage identification element when anelectrode applicator 312 is coupled to the instrument 300.

As an illustrative example, the following table maps resistor values tosetpoint voltages:

Needle Array Voltage ID Resistor (ohms) Setpoint Voltage 787  560 4531130 232 1500 1.32K  845 2.21K  845 1.29K 1300

The same or different identification elements may be used for typeidentification and voltage identification. The nature of theidentification element may vary as well. For example, an electroniccircuit may be incorporated into each electrode applicator 312 withstored digital or analog values for a variety of variables. Examples ofinformation that may be coded into an electrode applicator 312 are:needle array type parameters, such as number of needles, needle spacing,needle array geometry, and/or needle switching sequence; electricalpulse parameters such as voltage setpoint, pulse length, and/or pulseshape; shelf life; and usage limit. If the electrode applicator 312 usesa writable active circuit which can store data (e.g., an NVRAM), otherinformation which can be coded into an electrode applicator 312 include:shelf life lockout (i.e., a code that disables use of an electrodeapplicator 312 if its shelf life has expired); a usage count and lockout(i.e., a code that disables use of an electrode applicator 312 if thenumber of allowed uses has been reached; when an electrode applicator312 is designed to be disposable, this feature prevents contaminationfrom re-use); usage history (e.g., a log which records the number ofpulses applied, date and time of application, etc.); and error codecapture (e.g., to allow an electrode applicator 312 to be returned tothe manufacturer and analyzed for failure modes of the applicator or ofthe instrument 300).

The lockout may be determined by the length of time from initial use ofthe applicator as well as the number of therapy applications from asingle device. This may be accomplished by writing a time stamp to thedisposable applicator “key” element active circuit upon initialconnection to the instrument and would not allow use beyond a certainlength of time afterward. The time length limitation would be determinedby the maximum practical time length of one surgical procedure.

Furthermore, the usage of the “key” element may include manufacturingand quality control information. One example of such information is lotcode of the device. Also, it may aid in the quality control of thedevice by not allowing untested material to be used, e.g., the device isconfigured for use only after it has successfully completed amanufacturing test inspection.

Laparoscopic Needle Applicator

One embodiment of the invention that is particularly useful for treatinginternal tumors combines a laparoscopic needle array and the endoscopicexamination system to permit minimally invasive EPT. FIG. 8 is a diagramof a prior art endoscopic examination system 800. Light from a lightsource 840 is transmitted through a fiber optic light guide 842 to anendoscope 844, in known fashion. Tissue is illuminated from lightemanating from the distal end of the endoscope 844. Reflected light isgathered by the distal end of the endoscope 844 and transmitted to aneyepiece 846 or to a video camera 848 via an optical coupler 850. Asignal from the video camera 848 may be recorded on a video cassetterecorder 852 and/or displayed on a video monitor 854.

FIGS. 9 a-9 b are partially phantom side views of the distal end of animprovement over the endoscope 844 of FIG. 8, showing in detail anextending/retracting needle array 960 in accordance with the invention.A movable sheath 962 encloses an endoscope 944 and the needle array 960.FIG. 9 a shows the sheath 262 in an extended position, fully coveringthe endoscope 944 and the needle array 960. FIG. 9 b shows the sheath962 in a retracted position, exposing the distal ends of the endoscope944 and the needle array 960. (While the preferred embodiment uses amovable sheath 962, all that is required is relative movement betweenthe sheath 962 and the endoscope 944; hence, the endoscope 944 may beregarded as the movable element.)

In the preferred embodiment, the needle array 960 includes at least twoelectrode needles 964, each coupled to a voltage supply (not shown), andat least one of which may be hollow and coupled via tubing 966 to a drugsupply (not shown). The tips of the electrode needles 964 are preferablypositioned to extend beyond the distal end of the endoscope 944, so thata tissue site can be viewed with the endoscope 944 while the electrodeneedles 964 are inserted into the tissue.

Each electrode needle 964 is coupled to a compressible mechanism 968. Inthe illustrated embodiment, the compressible mechanism 968 includes, foreach electrode needle 964, a support arm 970 pivotably coupled to aslidable base 972 that is free to move along the endoscope 944, and to aprimary extension arm 974. Each primary extension arm 974 is pivotablycoupled to a fixed base 976 that is attached to the endoscope 944, andto a corresponding electrode needle 964. A secondary extension arm 977,similar in construction to the primary extension arm 974 (but without asupport arm 970) is provided for added stability of the electrodeneedles 964 when in a deployed configuration, described below.

When the sheath 962 is in an extended position, the electrode needles964 are in relatively close proximity to each other. While in some usesthis degree of proximity may be adequate for particular voltages, inother uses the electrode needles 964 need to have greater separation.

Accordingly, in the preferred embodiment, when the sheath 962 is movedto the retracted position, a compression element 978 (e.g., a spring)biases each slidable base 972 away from the fixed base 976, causing eachsupport arm 970 to pull on the coupled primary extension arm 974. Thisretractive force causes the extension arms 974, 977 to angle out fromthe endoscope 944 into a deployed configuration, thus increasing theseparation between the electrode needles 964 as shown in FIG. 9 b.

When the sheath 962 is moved to the extended position, the sheath 962compresses the electrode needles 964 together, forcing the extensionarms 974, 977 to fold. This causes each primary extension arm 974 topull on the coupled support arm 970. The retractive force on eachsupport arm 970 causes each slidable base 972 to move towards the fixedbase 976 into a sheathed configuration, compressing the compressionelement 978, as shown in FIG. 9 a.

Other compressible mechanisms 968 may be used to separate the electrodeneedles 964, as such as wedges (or a hollow core cone) of compressibleelastomeric material (such as foam or rubber) lodged between theendoscope 944 and the electrode needles 964, such that the widestportion of the wedges are at the distal end of the endoscope 944. Whenthe sheath 962 is in an retracted position, the elastomeric materialexpands more at the distal end of the wedges than at the proximal end ofthe wedges, thus increasing the separation between the electrode needles964. Further, not every electrode needle 964 need be movable by acompressible mechanism 968. For example, sufficient separation betweentwo electrode needles 964 may be achieved if one of the electrodeneedles 964 is held in a fixed position relative to the endoscope 944while the other electrode needle 964 is movable between a compressed andextended position; the two electrode needles 964 would be asymmetricallydisposed with respect to the endoscope 944 when in a deployedconfiguration.

In any case, the compressible mechanism 968 must provide electricalisolation between each electrode needle 964, and thus is preferably madein whole or in part of a dielectric such as non-conductive plastic.

While the preferred embodiment of a laparoscopic needle array includesan endoscope, in some embodiments it may be useful to use thelaparoscopic needle array with a separate endoscope. In thisconfiguration, a support rod can be substituted in FIGS. 15 a and 15 bfor the endoscope 944.

Electric Field Parameters

The nature of the electric field to be generated is determined by thenature of the tissue, the size of the selected tissue and its location.It is desirable that the field be as homogenous as possible and of thecorrect amplitude. Excessive field strength results in lysing of cells,whereas a low field strength results in reduced efficacy. The electrodesmay be mounted and manipulated in many ways including but not limited tothose in the parent application. The electrodes may be convenientlymanipulated on and by forceps to internal position.

The waveform of the electrical signal provided by the pulse generatorcan be an exponentially decaying pulse, a square pulse, a unipolaroscillating pulse train, a bipolar oscillating pulse train, or acombination of any of these forms. The nominal electric field strengthcan be from about 10 V/cm to about 20 kV/cm (the nominal electric fieldstrength is determined by computing the voltage between electrodeneedles divided by the distance between the needles). The pulse lengthcan be about 10 μs to about 100 ms. There can be any desired number ofpulses, typically one to 100 pulses per second. The wait between pulsessets can be any desired time, such as one second. The waveform, electricfield strength and pulse duration may also depend upon the type of cellsand the type of molecules that are to enter the cells viaelectroporation.

The various parameters including electric field strengths required forthe electroporation of any known cell is generally available from themany research papers reporting on the subject, as well as from adatabase maintained by GENETRONICS, INC., San Diego, Calif., assignee ofthe subject application. The electric fields needed for in vivo cellelectroporation, such as EPT, are generally similar in magnitude to thefields required for cells in vitro. Recent investigation by theinventors show that the preferred magnitudes are in the range of from 10V/cm to about 1300 V/cm. The higher end of this range, over about 600V/cm, has been verified by in vivo experiments of others reported inscientific publications.

The nominal electric field can be designated either “high” or “low”.Preferably, when high fields are used, the nominal electric field isfrom about 700 V/cm to 1300 V/cm and preferably from about 1000 V/cm to1300 V/cm. Preferably, when low fields are used, the nominal electricfield is from about 10 V/cm to 100 V/cm, and more preferably from about25 V/cm to 75 V/cm. In a particular embodiment, it is preferred thatwhen the electric field is low, the pulse length is long. For example,when the nominal electric field is about 25-75 V/cm, it is preferredthat the pulse length is about 10 msec.

Preferably, the therapeutic method of the invention utilizes theapparatus of the invention which provides an electrode apparatus for theapplication of electroporation to a portion of the body of a patientcomprises a support member, a plurality of needle electrodes mounted onsaid support member for insertion into tissue at selected positions anddistances from one another, and means including a signal generatorresponsive to said distance signal for applying an electric signal tothe electrodes proportionate to the distance between said electrodes forgenerating an electric field of a predetermined strength.

Alternatively, it is understood that other systems could be utilized inthe therapeutic method of the invention (e.g., for low voltage, longpulse treatment), for example, a square wave pulse electroporationsystem. For example, the ElectroSquarePorator (T820), available fromGENETRONICS, INC. of San Diego, Calif., U.S.A., can be used. Square waveelectroporation systems deliver controlled electric pulses that risequickly to a set voltage, stay at that level for a set length of time(pulse length), and then quickly drop to zero. This type of systemyields better transformation efficiency for the electroporation of plantprotoplast and mammalian cell lines than an exponential decay system.

The ElectroSquarePorator (T820) is the first commercially availablesquare wave electroporation system capable of generating up to 3000Volts. The pulse length can be adjusted from 5 μsec to 99 msec. Thesquare wave electroporation pulses have a gentler effect on the cellswhich results in higher cell viability.

The T820 ElectroSquarePorator is active in both the High Voltage Mode(HVM) (100-3000 Volts) and the Low Voltage Mode (LVM) (10-500 Volts).The pulse length for LVM is about 0.3 to 99 msec and for HVM, 5 to 99μsec. The T820 has multiple pulsing capability from about 1 to 99pulses.

Therapeutic Method

The therapeutic method of the invention includes electrotherapy, alsoreferred to herein as electroporation therapy (EPT), using the apparatusof the invention for the delivery of macromolecules to a cell or tissue.As described earlier, the term “macromolecule” or “molecule” as usedherein refers to drugs (e.g., chemotherapeutic agents), nucleic acids(e.g., polynucleotides), peptides and polypeptides, includingantibodies. The term polynucleotides include DNA, cDNA and RNAsequences.

Drugs contemplated for use in the method of the invention are typicallychemotherapeutic agents having an antitumor or cytotoxic effect. Suchdrugs or agents include bleomycin, neocarcinostatin, suramin,doxorubicin, carboplatin, taxol, mitomycin C and cisplatin. Otherchemotherapeutic agents will be known to those of skill in the art (seefor example The Merck Index). In addition, agents that are“membrane-acting” agents are also included in the method of theinvention. These agents may also be agents as listed above, oralternatively, agents which act primarily by damaging the cell membrane.Examples of membrane-acting agents include N-alkylmelamide andpara-chloro mercury benzoate. The chemical composition of the agent willdictate the most appropriate time to administer the agent in relation tothe administration of the electric pulse. For example, while not wantingto be bound by a particular theory, it is believed that a drug having alow isoelectric point (e.g., neocarcinostatin, IEP=3.78), would likelybe more effective if administered post-electroporation in order to avoidelectrostatic interaction of the highly charged drug within the field.Further, such drugs as bleomycin, which have a very negative log P, (Pbeing the partition coefficient between octanol and water), are verylarge in size (MW=1400), and are hydrophilic, thereby associatingclosely with the lipid membrane, diffuse very slowly into a tumor celland are typically administered prior to or substantially simultaneouswith the electric pulse. In addition, certain agents may requiremodification in order to allow more efficient entry into the cell. Forexample, an agent such as taxol can be modified to increase solubilityin water which would allow more efficient entry into the cell.

Electroporation facilitates entry of bleomycin or other similar drugsinto the tumor cell by creating pores in the cell membrane.

In one embodiment, the invention provides a method for the therapeuticapplication of electroporation to a tissue of a subject for introducingmolecules into cells therein, comprising providing an array ofelectrodes, at least one of the electrodes having a needle configurationfor penetrating tissue; inserting the needle electrode into selectedtissue for introducing molecules into the tissue; positioning a secondelectrode of the array of electrodes in conductive relation to theselected tissue; applying pulses of high amplitude electric signals tothe electrodes, proportionate to the distance between the electrodes,for electroporation of the tissue. It should be understood that theelectroporation of tissue can be performed in vitro, in vivo, or exvivo. Electroporation can also be performed utilizing single cells,e.g., single cell suspensions or in vitro or ex vivo in cell culture.

It may be desirable to modulate the expression of a gene in a cell bythe introduction of a molecule by the method of the invention. The term“modulate” envisions the suppression of expression of a gene when it isover-expressed, or augmentation of expression when it isunder-expressed. Where a cell proliferative disorder is associated withthe expression of a gene, nucleic acid sequences that interfere with thegene's expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid, ribozymes, or triplexagents to block transcription or translation of a specific mRNA, eitherby masking that mRNA with an antisense nucleic acid or triplex agent, orby cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNA,since the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target cell. The use of antisensemethods to inhibit the in vitro translation of genes is well known inthe art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The invention also provides gene therapy for the treatment of cellproliferative or immunologic disorders mediated by a particular gene orabsence thereof. Such therapy would achieve its therapeutic effect byintroduction of a specific sense or antisense polynucleotide into cellshaving the disorder. Delivery of polynucleotides can be achieved using arecombinant expression vector such as a chimeric virus, or thepolynucleotide can be delivered as “naked” DNA for example.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). When the subject is a human, a vector such as thegibbon ape leukemia virus (GaLV) can be utilized. A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated.

Therapeutic peptides or polypeptides may also be included in thetherapeutic method of the invention. For example, immunomodulatoryagents and other biological response modifiers can be administered forincorporation by a cell. The term “biological response modifiers” ismeant to encompass substances which are involved in modifying the immuneresponse. Examples of immune response modifiers include such compoundsas lymphokines. Lymphokines include tumor necrosis factor, interleukins1, 2, and 3, lymphotoxin, macrophage activating factor, migrationinhibition factor, colony stimulating factor, and alpha-interferon,beta-interferon, and gamma-interferon and their subtypes.

Also included are polynucleotides which encode metabolic enzymes andproteins, including antiangiogenesis compounds, e.g., Factor VIII orFactor IX. The macromolecule of the invention also includes antibodymolecules. The term “antibody” as used herein is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′)₂.

Administration of a drug, polynucleotide or polypeptide, in the methodof the invention can be, for example, parenterally by injection, rapidinfusion, nasopharyngeal absorption, dermal absorption, and orally. Inthe case of a tumor, for example, a chemotherapeutic or other agent canbe administered locally, systemically or directly injected into thetumor. When a drug, for example, is administered directly into thetumor, it is advantageous to inject the drug in a “fanning” manner. Theterm “fanning” refers to administering the drug by changing thedirection of the needle as the drug is being injected or by multipleinjections in multiple directions like opening up of a hand fan, ratherthan as a bolus, in order to provide a greater distribution of drugthroughout the tumor. As compared with a volume that is typically usedin the art, it is desirable to increase the volume of thedrug-containing solution, when the drug is administered (e.g., injected)intratumorally, in order to insure adequate distribution of the drugthroughout the tumor. For example, in the EXAMPLES using mice herein,one of skill in the art typically injects 50 μl of drug-containingsolution, however, the results are greatly improved by increasing thevolume to 150 μl. In the human clinical studies, approximately 20 ml wasinjected to ensure adequate perfusion of the tumor. Preferably, theinjection should be done very slowly all around the base and by fanning.Although the interstitial pressure is very high at the center of thetumor, it is also a region where very often the tumor is necrotic.

Preferably, the molecule is administered substantially contemporaneouslywith the electroporation treatment. The term “substantiallycontemporaneously” means that the molecule and the electroporationtreatment are administered reasonably close together with respect totime. The administration of the molecule or therapeutic agent can at anyinterval, depending upon such factors, for example, as the nature of thetumor, the condition of the patient, the size and chemicalcharacteristics of the molecule and half-life of the molecule.

Preparations for parenteral administration include sterile or aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Besides the inert diluents, such compositions can alsoinclude adjuvants, wetting agents, emulsifying and suspending agents.Further, vasoconstrictor agents can be used to keep the therapeuticagent localized prior to pulsing.

Any cell can be treated by the method of the invention. The illustrativeexamples provided herein demonstrate the use of the method of theinvention for the treatment of tumor cells, e.g., pancreas, lung, headand neck, cutaneous and subcutaneous cancers. Other cell proliferativedisorders are amenable to treatment by the electroporation method of theinvention. The term “cell proliferative disorder” denotes malignant aswell as non-malignant cell populations which often appear to differ fromthe surrounding tissue both morphologically and genotypically. Malignantcells (i.e., tumors or cancer) develop as a result of a multi-stepprocess. The method of the invention is useful in treating malignanciesor other disorders of the various organ systems, particularly, forexample, cells in the pancreas, head and neck (e.g., larynx,nasopharynx, oropharynx, hypopharynx, lip, throat,) and lung, and alsoincluding cells of heart, kidney, muscle, breast, colon, prostate,thymus, testis, and ovary. Further, malignancies of the skin, such asbasal cell carcinoma or melanoma can also be treated by the therapeuticmethod of the invention (see Example 2). Preferably the subject ishuman, however, it should be understood that the invention is alsouseful for veterinary uses in non-human animals or mammals.

In yet another embodiment, the invention provides method for thetherapeutic application of electroporation to a tissue of a subject fordamaging or killing cells therein. The method includes providing anarray of electrodes; positioning a second electrode of the array ofelectrodes in conductive relation to the selected tissue; and applyingpulses of high amplitude electric signals to the electrodes,proportionate to the distance between the electrodes, forelectroporation of the tissue. The method preferably utilizes lowvoltage and a long pulse length which precludes the need for additionalcytotoxic or chemotherapeutic agents. For example, preferably thenominal electric field is from about 25 V/cm to 75 V/cm and the pulselength is from about 5 μsec to 99 msec.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

The following examples illustrate the use of EPT in cell lines, animalsand humans. Example 1 illustrates EPT in poorly differentiated humanpancreatic tumors (Panc-3) xenografted subcutaneously on the flank ofnude mice. Example 2 shows the results of clinical trials in humansusing EPT for treatment of basal cell carcinomas and melanomas. Example3 shows results of clinical trials in humans using EPT for treatment ofhead and neck tumors. Example 4 provides in vitro data for EPT utilizinglow voltage (electric field) and long pulse length. The parameters forEPT are described in the examples; for Example 1 and for the head andneck clinical trials, the nominal electric field was 1300 V/cm and 6pulses for 99-100 μsec, spaced at 1 second intervals. The clinicaltrials (Example 2) used similar parameters, however the electric fieldwas 1130 V/cm. (Nominal electric field (V/cm) is applied voltage (V)across the needle pairs divided by the distance between the needle pairs(cm).) The Examples illustrate the use of EPT for effectively killingundesired cell populations (e.g., a tumor) in vitro and in vivo.

Example 1 EPT for Treatment of Tumors in vivo

The single treatment procedure involved injection of bleomycin (0.5units in 0.15 ml saline) intratumorally, using fanning, as describedherein followed by application of six square wave electrical pulses, tenminutes later, using needle array electrodes as described in the presentapplication, arranged along the circumference of a circle 1 cm indiameter. Needle array of variable diameters (e.g., 0.5 cm, 0.75 cm and1.5 cm can also be used to accommodate tumors of various sizes. Stoppersof various heights can be inserted at the center of the array to makethe penetration depth of the needles into the tumor variable. A built-inmechanism allowed switching of electrodes for maximum coverage of thetumor by the pulsed field. The electrical parameters were: 780 V/cmcenter field strength and 6×99 μs pulses spaced at 1 sec interval.

Results showed severe necrosis and edema in nearly all the mice at thetreatment site. While there was a substantial reduction in the tumorvolume (after a slight initial increase due to edema) of the mice in thetreated group (D+E+; D=Drug, E=Electrical field), those in the controlgroup (D+E−) increased dramatically. Histological analysis of tumorsamples showed necrotic tumor cell ghosts in D+E+ group compared to amixture of viable and necrotic cells in D+E− group. Preliminary studieswith human non-small cell lung cancer (NSCLC) tumors xenografted ontonude mice have also shown very encouraging results with EPT treatmentwith bleomycin.

The tumor cell line Panc-3, a poorly differentiated adenocarcinoma cellline of the pancreas, was supplied by AntiCancer, Inc., San Diego. ForEPT experiments, tissue taken from the stock mice, where the tumor linewas maintained, was thawed and cut into very small pieces about 1 mmeach, and 8-10 pieces were surgically xenografted in a subcutaneous sacmade in left flank of nude mice, and then closed with 6.0 surgicalsuture. After the average tumor size reached about 5 mm, mice withpalpable tumors were divided randomly, 10 mice for control group (D+E−;D=Drug, E=Electric field) and 10 mice for EPT treatment, namelybleomycin injection followed by pulsing (D+E+) from a BTX Square WaveT820 Generator. The tumor dimensions were measured and the tumor volumecalculated using the formula:(II/6)×a×b×cwhere a, b, and c a are, respectively, the length, width and thicknessof the tumor. 0.5 units Bleomycin (Sigma Chemicals) was dissolved in0.15 ml of 0.9% NaCl and was injected in each mice intratumorally byfanning for both the control (D+E−) and the treated (D+E+) groups. Tenminutes after the injection, each mouse in the D+E+ group was pulsedfrom a BTX T820 square wave electroporator with a set of needle arrayelectrodes as described in the present invention. Electrical parametersused were as follows: field strength 1300 V/cm, 6 pulses of 99 μs each,at 1 sec interval.

The mice were monitored every day for mortality and any signs of adiseased state were noted. The tumor dimensions were measured at regularintervals and tumor growth regression/progression monitored.

FIG. 10 shows the EPT results of various control and treated animalswith and without drug and/or with and without pulsing using bleomycinfor the Panc-3 tumors. There was a dramatic difference between theuntreated and treated mice in terms of tumor volume. There wasessentially no detectable tumor after approximately 24 days treatment.The results of FIG. 10 are also summarized in Table 1 below up to 43days. An illustration of the actual regression of the tumor is shown inthe sequence of FIGS. 13 a-13 d and the corresponding histology in FIGS.14 a-14 c.

TABLE 1 ELECTROCHEMOTHERAPY OF PANC-3 TUMORS IN NUDE MICE Tumor TumorTumor Tumor Days after volume volume volume volume treatment (mm³) C1(mm³) C2 (mm³) T1 (mm³) T2  0 138.746 148.940 123.110 178.370  1 206.979179.820 210.950 252.720  8 394.786 451.787 104.550 211.110 15 557.349798.919 113.210 226.966 18 939.582 881.752 161.730 246.910 24 1391.0571406.980 41.560 47.223 28 1628.631 1474.210 0 0 35 2619.765 2330.310 0 038 2908.912 2333.967 0 0 43 3708.571 5381.759 0 0 Cell Line: poorlydifferentiated human pancreatic tumor (Panc-3) Mouse model: nude mouseTransplant: subcutaneous xenograft Control mice: C1 and C2 Treated mice:T1 and T2

The Panc-3 experiment was repeated using a non-small cell lung cancercell line (NSCLC), 177 (AntiCancer, San Diego, Calif.). The results weresimilar to that found with bleomycin and Panc-3 as shown in FIG. 10. Inone experiment, a tumor had recurred was retreated at day 27 (FIG. 12)and after 7 days, there was no evidence of tumor.

The Panc-3 and NSCLC models were utilized with the drug neocarcinostatin(NCS) following the same procedures as outlined above. As shown in FIG.1 a, pre-pulse dosing with NCS in a manner similar to that used for thebleomycin studies, was not effective in reducing tumor size at all. Itwas believed that due to the low isoelectric point of NCS, electrostaticinteraction prevented the drug from entering the tumor cell. Therefore,the experiment was repeated by pulsing first and injecting NCSpost-pulse.

FIG. 11 b shows the initial tumor volume (I) as compared to the finaltumor volume (F) at day 13 for 7 mice treated (Mouse ID 1-7). In severalof the mice (ID 1, 2, 4, and 7), an increase in tumor volume wasobserved, but appeared to be due to edema. However, as shown in FIG. 20d, when a separate group of 5 mice were examined at day 23, all miceshowed a marked reduction in tumor volume.

A comparison of FIGS. 11 a and 11 b indicated that post-pulse with NCSwas more effective than pre-pulse administration for NCS.

The present Example illustrates that a poorly differentiated Pancreaticcancer (Panc-3) and Non-small cell lung cancer (NSCLC) xenograftedsubcutaneously onto nude mice can be effectively treated by the EPTmethod of the invention using bleomycin or NCS and needle arrayelectrodes. Other similar chemotherapeutic agents can also be effectiveusing the method of the invention.

The response of Panc-3 to ECT with bleomycin is shown in Table 2. In 68%(17/25) of the treated mice, complete tumor regression was observed 28days following treatment, while 20% (5/25) showed partial (>80%)regression, 8% (2/25) showed no response and 4% (1/25) died, 20 daysafter treatment. No palpable tumor was observed in 64% (16/25) of thecases even after 120 days of the treatment. Representative animals(2/17) from this group were monitored to be without tumors for 243 daysafter which these were humanely euthanized. In 8% of the mice, however,there was tumor regrowth 35 days after treatment, but at a much slowerrate.

Histological studies clearly showed severe necrosis of the tumor regionfor the group subjected to EPT whereas no necrosis was apparent in thecontrol group. Intratumoral drug injection with larger volume ofbleomycin, combined with fanning to maximize uniform drug distributionthroughout the tumor volume, was found to be very effective as comparedto the conventional mode of injecting the drug prior to pulsing.

TABLE 2 Electrochemotherapy of Panc-3 with Bleomycin Days aftertreatment Number of mice treated: 25 28 35 57 84 94 120 CompleteRegression (100%) 17  16  16  16  16  16 Partial Regression (≧80%) 5 3 33 3   3^(e) No Response 2 2  1^(a) 1 1 Death 1  2^(b) Tumor regrowth 2 1^(d) Retreatment 2 Histology 1 ^(a),^(c)Mice sacrificed due toincreased tumor burden ^(b)1 mouse died after treatment; 1 mouse with nopalpable tumor died after 64 days survival ^(d)Secondary metastatictumor ^(e)Fibrous tissueIn vivo Results Using the MedPulser™.

Preliminary experiments using MedPulser™ (apparatus of the invention)for treatment of tumor xenografts grown subcutaneously onto nude micehave shown encouraging results. Human pancreatic xenograft (Panc-4) whentreated with EPT using MedPulser™ and bleomycin showed complete tumorregression in about 75% of the mice treated up to day 39 of observation.Treatment of human prostate xenografts (PC-3) has also shown about 66%complete regression of tumors. (No tumors observed up to 60 days aftertreatment). Both 4 and 6 needle array are effective in treatment oftumors by EPT.

Comparison of MedPulser™ 4 and 6 Needle Array for in vitro Experimentswith PC-3

Experiments were carried out to compare the efficacy of 6 vs. 4 needlearray with MedPulser™ on PC-3 (human prostate cell line) in vitro. Cellswere suspended in RPMI media and seeded uniformly at 200,000 cells/ml.Bleomycin at 2×10⁵ M was added to the wells (for D+E− and D+E+ only).Cells were electroporated in 24 well plates using the 6-needle and4-needle array electrodes connected to the MedPulser. The electropulseparameters were 6×99 μs, 1129 V with the 6-needle array and 4×99 μs, 848V with the 4-needle array. The cells were transferred to 96 well platesand incubated for 20 hours at 37° C. The cell survival was determinedusing XTT assay which is based on metabolic conversion of XTT toformazan and is measured spectrophotometrically at 450 nm. Only thecells which are live convert XTT to formazan. The percent cell survivalvalues are relative values calculated from the O.D. values of thesample, a control with 100% cell survival (D−E−) and control with 0%cell survival (D−E− with SDS, which lyses all cells). The cell survivaldata are as follows:

TABLE 3 Treatment Avg. % Survival SE D − E − 100 3.65 (n = 6) D + E −27.71 1.05 (n = 6) D − E + (4N) 101.15 4.32 (n = 12) D − E + (6N) 97.724.33 (n = 12) D + E + (4N) 4.78 7.53 (n = 12) D + E + (6N) −4.12 0.59 (n= 12)

From the preliminary data obtained in the experiments, it can beconcluded that statistically both 4 and 6 needles appear to be equallyeffective in killing the tumor cells in vitro.

Example 2 Clinical Trials for Basal Cell Carcinomas and Melanomas

The effectiveness of bleomycin-EPT on the tumors was to be assessed bythe end of the eight-week period using the same tumor response criteriaas employed in Example 1.

The concentration of bleomycin administered was 5 U/1 mL. The dosages ofbleomycin were administered as follows:

TABLE 4 Tumor Size Dose of Bleomycin <100 mm³ 0.5 U 100-150 mm³ .75 U150-500 mm³ 1.0 U 500-1000 mm³ 1.5 U 1000-2000 mm³ 2.0 U 2000-3000 mm³2.5 U 3000-4000 mm³ 3.0 U ≧5000 mm³ 4.0 UTable 5, following, shows the results of the responses to treatment.

-   -   NE=no effect; less than 50% reduction in tumor volume.    -   PR=partial responses; 50% or greater reduction in tumor volume.    -   CR=complete response; disappearance of all evidence of tumor as        determined by physical examination, and/or biopsy.

TABLE 5 Subject Response to Treatment Tumor Total # NE PR CR TypeNodules D′E′ D′D DE′ DE D′E′ D′D DE′ DE D′E′ D′D DE′ DE TUMOR RESPONSEBCC 67 0/44 5/6 3/15 2/2 1/44 1/6 4/15 0/2 43/44 0/6 0/15 0/2 Mel 971/58 5/5 4/30 4/4 4/58 0/5 4/30 0/4 53/58 0/5 0/30 0/4 Other  8 0/5  3/30/0  0/0 1/5  0/3 0/0  0/0 4/5 0/3 0/0  0/0 Total 172   1/107 13/14 7/456/6  6/107  1/14 8/45 0/6 100/107  0/14 0/45 0/6 % RESPONSE BCC 67 0  8320 100 2 17  27 0 98 0 0 0 Mel 97 2 100 13 100 7 0 13 0 91 0 0 0 Other 8 0 100  0  0 20  0  0 0 80 0 0 0 Total 172  1  93 16 100 6 7 18 0 93 00 0

Example 3 EPT for Head and Neck Cancers

All of the following patients were treated with bleomycin intratumoralinjection and needle arrays of different diameters with six needles. Thevoltage was set to achieve a nominal electric field strength of 1300V/cm (the needle array diameter was multiplied by 1300 to give thevoltage the generator was set at). The pulse length was 100 μs.

Study Methods

The study was designed as a single center feasibility clinical study inwhich the efficacy of the EPT procedure in combination withintralesional bleomycin was compared to that for traditional surgery,radiation, and/or systemic chemotherapy. Approximately 50 study subjectswere enrolled in the study. All study subjects were assessed prior totreatment by examination and biopsy. Postoperative assessment of studysubjects was weekly for 4-6 weeks, and monthly thereafter for a total of12 months. Approximately 8 to 12 weeks following therapy, a biopsy ofthe tumor site was performed. Use of CT or MRI scans was utilized inaccordance to standard medical follow-up evaluation of HNC subjects.

Tumor evaluation includes measuring the tumor diameter (in centimeters)and estimating its volume (in cubic centimeters). Prior to intratumoraladministration of bleomycin sulfate, the tumor site is anesthetized with1% lidocaine (xylocaine) and 1:100,000 epinephrine. The concentration ofbleomycin sulfate injected is 4 units per milliliter, up to a maximumdose of 5 units per tumor. If more than one tumor per subject istreated, a total of 20 units per subject should not be exceeded. Thedose of bleomycin administered is to be 1 unit/cm³ of calculated tumorvolume. Approximately ten minutes subsequent to the injection ofbleomycin sulfate, the applicator is placed on the tumor and electricalpulses initiated. Each application or an initiation of electrical pulsesis referred to as a sequence. The use of EPT is not a contraindicationto any subsequent palliative treatment required by the subject.

In this study, success was defined as significant tumor regression in aperiod of 16 weeks or less without major side effects seen withtraditional therapy. There are three possible response outcomes:

Complete Response (CR): Disappearance of all evidence of tumor asdetermined by physical examination, and/or biopsy.

-   -   Partial Response (PR): 50% or greater reduction in tumor volume.    -   No Response (NR): less than 50% reduction in tumor volume.

If the tumor increases (25% tumor volume) in size, other therapy, ifindicated, was instituted per subject's desire.

Subject's Response to Treatment

Table 6 displays the subject's response to treatment. Three subjects hada complete response (Subject No. 1, 3 and 4); four subjects have had apartial response (Subject No. 2, 6, 8 and 9); and two subjects had noresponse (Subject No. 5 and 7) to treatment. Three subjects died priorto reaching week 12 due to progressive disease or complicationsunrelated to study treatment (Subject No. 2, 5 and 7). One of the threesubjects achieved a PR at week 4 (Subject No. 2). Two subjects had noprevious clinical cancer treatments for their tumor prior to studyenrollment (Subject No. 4 and 8). Three subjects had a tumor that wasnot completely accessible to the applicator component of the device andtherefore received segmented treatment (Subject No. 5, 7 and 9).

Table 7 shows a summary of clinical studies using bleomycin sulfate andEPT using the apparatus of the invention, MedPulser™.

TABLE 6 Response to Bleomycin Sulfate/EPT Time to Subject Previous Weekof Response Response Last Visit No./Initials Treatment Treatment (Week)Status (Week) 1/J-S S 0 2, 8 PR, CR 22 2/G-C R 0, 4 4 PR 4 3/L-O R 0 3CR 16 4/G-R None 0, 4 4, 9 PR, CR 9 5/R-H R 0, 4 na NR** 4 6/C-B R 0, 122 PR 12 7/C-J S, R, C 0 na NR** 1 8/L-J None 0, 6 4 PR 9 9/J-T S, R, C0.7 7 PR** 7 (S) Surgery (R) Radiation (C) Chemotherapy PR—PartialResponse CR—Complete Response NR—No Response **Segmented treatment

TABLE 7 Summary of Clinical Studies Using Bleomycin Sulfate andElectroporation Therapy EPT Tumor Response No. of Tumor No of Bleomycin(Units) (Sequences) CR PR NR NE Site/P.I. Status Patients HistologyVolume Lesions IT IV TOTAL (n) % (n) % (n) % (n) Head and Enrollment 7Squamous 0.52- 7  1 to 16  1-14 3 43 2 29 2 29  2 Neck Start: 08/14/96Cell 25.12 cm³ Cancer Stop: 18/18/96 Status: Patients in Follow-Up 2Adeno- 4.12- 2 2 to 8 2-8 0  0 2 100  0 100  carcinoma 12.56 cm³Cutaneous Summary 9 9 3 33% 4 44% 2 22%  2 and Sub- Enrollment 7 BasalCell 0.07- 8  0.8 to 0.64 1 4 67 2 33 0 0 cutaneous Start: 08/01/96 0.63cm³ Cancer Stop: 01/27/97 Status: 5 Patients in Follow-Up 2 Patients tobe evaluated Cutaneous Summary 7 8 4 67% 2 33% 0 0% 2 and Sub-Enrollment 18 Basal Cell 0.019- 54 0.5 to 3   1-5 51 94 5  9 0 0cutaneous Start: 01/20/95 2.023 cm³ Cancer Stop: 11/05/96 Status:Completed 10 Met. 0.07- 84 0.2 to 2   1-8 75 91 8  9 2 2 Melanoma 5.376cm³ 1 Kaposi 0.014- 4 1 to 2 1-2 4 100  0  0 0 0 Sarcoma 4.472 cm³ 1Squamous 0.453 cm³ 1 1.5 1-4 0  0 1 100  0 0 Cell Cutaneous Summary 30146 130 89% 14  10% 2 1% and Sub- Enrollment 2 Basal Cell 0.078- 6 22 to23 1 1 17 5 83 0 0 cutaneous Start: 02/18/94 0.782 cm³ Cancer Stop:05/04/94 Status: Completed 3 Met. 0.038- 10 19 to 23 1 3 30 2 20 5 50 Melanoma 1.786 cm³ 1 Met. Breast 0.285- 2 17 1 2 100  0  0 0 0 Adeno-0.454 cm³ carcinoma CR = Complete Response PR = Partial Response NR = NoResponse NE = To be evaluated for response

Example 4 Low Voltage Long Pulse Length (LVLP) EPT

Conventional electrochemotherapy uses high voltages short pulsedurations for treatment of tumors. The electrical field conditions of1200-1300 V/cm and 100 μs have been found to be very effective in vitroand in vivo with anticancer drugs like bleomycin, cisplatin, peplomycin,mitomycin c and carboplatin. These results refer to in vitro and in vivowork. Although such electrical conditions are well tolerated by patientsin clinical situations, such treatments will typically produce muscletwitch and occasional discomfort to patients. The sensation ofdiscomfort is often found to be associated with individual patient'sperception of pain. Often patients respond very differently under thesame experimental conditions. Some of these problems could beconsiderably reduced by using low voltage high pulse durations forelectrochemotherapy. The lowest field strength reported for in vivo genetransfer is 600 V/cm (T. Nishi et al. Cancer Res. 56:1050-1055, 1996).The maximum field strength used for the in vitro EPT experiments areshown in Table 8 where the field strength necessary to kill 50% of thecells is <50V/cm.

The following in vitro experiments with various tumor cell lines, suchas MCF-7 (human breast cancer), PC-3 (human prostate cancer) and C6 (RatGlioma) have shown that low voltage, long pulse durations are equal orbetter than high voltage short pulse durations in terms of tumor cellkilling. Results are illustrated within MCF-7. Titration of pulse lengthhas shown that it can range from 4-15 msec. The electroporation responseof MCF-7 has been carried out at both high voltage/short pulse length(HVSP) and low voltage/long pulse length (LVLP) using an XTT assay after70 hours which is based on metabolic conversion of XTT to formazan whichis measured spectrophotometrically at 450 nm. (M. W. Roehm, et al., AnImproved Colorimetric Assay for Cell Proliferation and ViabilityUtilizing the Tetrazolium Salt XTT, J. Immunol. Methods 142:2, 257-265,1991.) XTT is a tetrazolium reagent,2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), which is metabolically reducedin viable cells to a water-soluble formazan product. Therefore, only thecells which are live convert XTT to formazan. The percent cell survivalvalues are relative values calculated using a formula from the O.D.values of the sample. (Control, with 100% cell survival (D-E) andcontrol with 0% cell survival (D-E with SDS)). The experiments with HVSPwere done to permit direct comparison with the currently developed LVLPmode of EPT.

TABLE 8 HVSP LVLP Cell line Cell Type LD₅₀ (V/cm) LD₅₀ (V/cm) MCF-7Breast Cancer (Human) 1800 50

Voltages as low as 25 V/cm caused significant cytotoxicity to the cells.An increase in the electric field resulted in complete cell killing.Some of the cell lines like C6 glioma which were not affected verysignificantly by high voltage pulses but were completely killed by lowvoltages of 20-30 V/cm. These in vitro results clearly establish thepotential of using the LVLP modality of EPT treatment.

Cytotoxicity of Drugs with EPT in vitro

Experimental results of in vitro EPT experiments with various drugsusing MCF-7 both high voltage and low voltage are described below.

Cells were obtained from ATCC (American Type Tissue Collection,Rockville, Md., U.S.A.) and maintained by their recommended procedures.Cells were suspended in appropriate medium and were uniformly seeded in24/96 well plates. One of the following drugs: bleomycin, cisplatin,mitomycin C, doxorubicin and taxol was added directly to the cellsuspensions at final concentrations of about 1×10⁻⁴ (1E-4) to 1.3×10−⁹(1.3E-9). The electrical pulse a BTX T820 electro square porator weredelivered to the cell suspensions in microplates using a BTX needlearray electrode as described herein. Depending on the experiment, sixpulses of either 100 μs or 10 ms and at various nominal electric fieldsof either high voltage or low voltages were applied between two oppositepairs of a six-needle array using EPT-196 needle array switch. Themicroplates were incubated for either 20 hrs or 70 hrs and the cellsurvival was measured by the XTT assay. Some of the results arepresented in FIGS. 15(a), 15(b), 16(a), 16(b) and 17.

The curves corresponding to FIG. 17 were obtained using the MedPulser™.

For the LVLP mode, the method shows that cell survivability is wellbelow 50% even when the cells are pulsed in the absence of drugs; thispercentage is further reduced when combined with the drugs. It is moredesirable to show that the drugs show the effect rather than the pulseand requires selecting initial survival values with the pulse alone atabout 80%. Typical cell killing curves for LVLP mode are shown in FIG.15(a).

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A method for the introduction of an agent into cells of a tissue,said method comprising: a) introducing the agent into tissue; and b)applying at least one voltage pulse between a plurality of opposingpairs of needle electrodes disposed in the tissue so as to establish anelectric field in cells of the tissue sufficient to cause electroportionof cells in the tissue, thereby introducing said agent into the cells ofthe tissue.
 2. The method of claim 1, wherein the agent is introduced byinjected either prior to, simultaneously with, or after step (b).
 3. Themethod of claim 2, wherein the agent is injected locally into thetissue.
 4. The method of claim 3, wherein the voltage pulses are appliedto more than two pairs of said electrodes disposed in the tissue.
 5. Themethod of claim 1, wherein said method is in vivo.
 6. The method ofclaim 1, wherein the agent is selected from the group consisting ofdrugs, nucleic acids, polynucleotides, chemotherapeutic agents,peptides, polypeptides, and antibodies.
 7. The method of claim 6,wherein the agent is a polynucleotide selected from the group consistingof a DNA, a cDNA, and an RNA.
 8. The method of claim 7, wherein thepolynucleotide is an antisense nucleic acid or ribozyme.
 9. The methodof claim 7, wherein the polynucleotide encodes a protein selected fromthe group consisting of an immunomodulatory agent, a biological responsemodifier, a metabolic enzyme, and an antiangiogenesis compound.
 10. Themethod of claim 7, wherein the polynucleotide is contained in a viralvector.
 11. The method of claim 10, wherein the viral vector is selectedfrom the group consisting of an adenovirus, a herpes virus, a vacciniavirus, and a retrovirus.
 12. The method of claim 11, wherein the viralvector is a retroviral vector that is a derivative of a murine or avianretrovirus.
 13. The method of claim 1, wherein the agent is achemotherapeutic agent.
 14. The method of claim 13, wherein saidchemotherapeutic agent is selected from the group consisting ofbleomycin, neocarcinostatin, suramin, doxorubicin, carboplatin, taxol,mitomycin C, and cisplatin.
 15. The method of claim 1, wherein the cellsare tumor cells.
 16. The method of claim 15, wherein the cells aremelanoma or basal cell carcinoma cells.
 17. The method of claim 16,wherein the tumor cells are subsurface tumor cells.
 18. The method ofclaim 1, wherein said tissue is mammalian.
 19. The method of claim 1,wherein said tissue is human.
 20. The method of claim 1, wherein saidelectrodes are contained in an array selected from the group consistingof a four needle, a six needle, an eight needle, a ten needle, a twelveneedle, a fourteen needle, and a sixteen needle array of electrodes. 21.The method of claim 1, wherein the electric field is from about 10 V/cmto about 2000 V/cm.
 22. The method of claim 1, wherein from about 1 toabout 100 electrical pulses are applied.
 23. The method of claim 22,wherein each electrical pulse is from about 10 microsecs to about 100msec in duration.
 24. The method of claim 1, wherein at least oneelectrical pulse is selected from the group consisting of a square wavepulse, an exponential wave pulse, a unipolar oscillating wave form, anda bipolar oscillating wave form.
 25. The method of claim 24, whereineach electrical pulse is comprised of a square wave pulse.
 26. A methodfor the introduction of an agent into cells of a tissue, said methodcomprising: a) introducing the agent locally into tissue by a routeother than dermal absorption; and b) applying voltage pulses to opposingpairs of electrodes disposed in the tissue so as to establish anelectric field in the tissue sufficient to cause the agent to entercells of the tissue, thereby introducing said agent into cells of thetissue.
 27. The method of claim 26, wherein said electrical pulse iscomprised of a square wave pulse.
 28. The method of claim 26, whereinthe route comprises rapid infusion.
 29. The method of claim 26, whereinthe route comprises nasopharyngeal absorption.
 30. The method of claim26, wherein the route comprises oral administration.
 31. A method ofelectroporating an agent into cells of a tissue, comprising: a)introducing a therapeutic agent into a tissue of a patient in need oftreatment; and b) using an electrode apparatus placed in contact withthe tissue to deliver voltage pulses that establish electric fieldssufficient to introduce the therapeutic agent into cells of the tissueby way of electroporation, wherein the electrode apparatus comprises: i.a support member having disposed thereon two or more opposing pairs ofneedle electrodes arranged relative to one another to form an electrodearray; and ii. a power supply in electrical communication with pairs ofneedle electrodes disposed in the support member, wherein the powersupply provides voltage pulses to at least two of the opposing pairs ofneedle electrodes to effect electroporation.